Membrane fusion provides a conceptually simple mechanism for enveloped viruses to deliver their genomes into the cytoplasm of target cells. In contrast, the mechanisms used by nonenveloped viruses to penetrate cellular membranes and release their genomes into the cell remain poorly understood. This application proposes a program of structural studies of poliovirus cell entry intermediates as a simple model for understanding how nonenveloped viruses gain entry into the cell. The problem is framed in the context of several specific questions: 1) What is the nature of the virus-receptor interaction and how is receptor binding coupled to the induction of conformational changes? 2) What is the sequence of conformational changes that ultimately leads to cell entry and the release of the viral RNA? 3) What is the nature of the interaction of the virus with the cell membrane that leads to the internalization of the RNA? 4) How is the RNA released from the particle and into the cell? From a structural perspective, these questions cover over six orders of magnitude in scale from the atomic level (tenths of nanometers) to the cellular level (tens of microns). This range is well beyond the scope of any one structural method. The project seeks to develop a hybrid approach combining x-ray crystallography, cryoelectron microscopy, cyroelectron tomography and optical microscopy to develop a series of structural snapshots of cell entry intermediates of poliovirus; and to use these structures to address questions posed by, and pose questions for, supporting biochemical and genetic studies. The specific aims include extending structural studies of soluble and membrane-associated poliovirus cell entry intermediates in vitro and initiating studies using a combination of fluorescence microscopy and electron tomography to probe the location, environment and roles of entry intermediates in cells. The goal will be to combine the high-resolution structures derived from the in vitro studies with information about the location of intracellular intermediates to define structural changes associated with cell entry and delivery of the genome into the cytoplasm in the context of the intact cell at an unprecedented level of detail. The information derived from these studies will be directly relevant to understanding the detailed cell entry mechanism of a group of viruses (the enteroviruses and rhinoviruses) that includes a number of significant human pathogens, and will provide important insights into the more general question of how nonenveloped viruses enter cells. The structural methods developed in these studies will also be generally useful to a wide range of questions in cell biology. PUBLIC HEALTH RELEVANCE: The project seeks to understand how poliovirus and related viruses such as rhinoviruses, coxsackieviruses, and enteroviruses (all of which are significant human pathogens) enter cells. The findings are also expected to be more generally relevant to many other viruses which lack a lipid envelop. Understanding of the viral entry pathways could lead to the development of novel antivirals.